Peptide spectral analysis

The integration of SpC data with mass ion intensity information can provide a more accurate indication of abundance (98). Similarly, methods weighting SpC data with data MSI feature extracted ion intensities improves reliability (99). To absolutely quantify proteins requires integration of the individual peptide spectral ions generated during mass-spectrometric analysis. To do this requires extraction of peptide MS2 spectra to obtain ion intensity and masses. By correlating ion intensity and accurate mass with internal standard spikes... 

Fig. 1. Structural representations and fidelity of synthesis of lipopeptide and nonlipidated peptides. (A) Structure of the lipopeptide vaccines palmitic acid, Pam. (B) HPLC chromatograms of purified lipopeptides. Analysis by HPLC was performed on a Waters HPLC System using a Vydac C4 column with 0.1% TFA in water and 0.1% TFA in acetonitrile as a gradient mobile phase. A flow rate of 1 mL/min was used at a gradient of 2%/min. Chromatograms were obtained by the detection of absorbance at a wavelength of 214 nm. (C) Mass spectral analysis of purified lipopeptides. Lipopeptides were analyzed by mass spectrometry using an Agilent 1100 Series LCM/MSD ion trap.

The need for a reverse-field polarity power supply is at least two-fold 1) It permit a complete spectral analysis of the substance under study. By reversing field polarity, the substance zones can be run forward and backward in front of the detector as many times as needed. Incremental changes as small as 1- or 2-nm in wavelength can be used to maximize instrumental sensitivity, thus allowing coverage of the entire spectral range. In fact, this feature provides the same functions as a diode array detector, albeit somewhat slower. Proteins and peptides have almost identical spectral characteristics, however, when other functional groups are attached to it, is possible to observe more than one maximal absorbance peak. For example,... 

Mass Spectral Analysis of Tryptic Map Peptides of Human Relaxin B-chain... 

S spectral analysis of the intact peptides (2) or their chymotryptic... 

Initial sequencing attempts with HPLC-purified Acheta PDCF indicated a blocked amino-terminus. It was deblocked by pyroglutamyl amino peptidase and subjected to gas-phase sequencing. The resulting data, along with spectral analysis, indicated that the purified peptide has the sequence pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-amide. This was also the deduced sequence for Gryllus AKH (43). The synthetic peptide. 

The particle beam LC/FT-IR spectrometry interface can also be used for peptide and protein HPLC experiments to provide another degree of structural characterization that is not possible with other detection techniques. Infrared absorption is sensitive to both specific amino acid functionalities and secondary structure. (5, 6) Secondary structure information is contained in the amide I, II, and III absorption bands which arise from delocalized vibrations of the peptide backbone. (7) The amide I band is recognized as the most structurally sensitive of the amide bands. The amide I band in proteins is intrinsically broad as it is composed of multiple underlying absorption bands due to the presence of multiple secondary structure elements. Infrared analysis provides secondary structure details for proteins, while for peptides, residual secondary structure details and amino acid functionalities can be observed. The particle beam (PB) LC/FT-IR spectrometry interface is a low temperature and pressure solvent elimination apparatus which serves to restrict the conformational motions of a protein while in flight. (8,12) The desolvated protein is deposited on an infrared transparent substrate and analyzed with the use of an FT-IR microscope. The PB LC/FT-IR spectrometric technique is an off-line method in that the spectral analysis is conducted after chromatographic analysis. It has been demonstrated that desolvated proteins retain the conformation that they possessed prior to introduction into the PB interface. (8) The ability of the particle beam to determine the conformational state of chromatographically analyzed proteins has recently been demonstrated. (9, 10) As with the ESI interface, the low flow rates required with the use of narrow- or microbore HPLC columns are compatible with the PB interface. 

B. Mass Spectral Analysis of the Active Site Peptides... 

Ion spray mass spectral analysis of peptides was performed by Peptidogenic Research, Livermore, CA. 

A third approach, pioneered by the group of Liebler [19, 34], involves a pattern recognition software called scoring algorithm for spectral analysis (SALSA) to search specific sequence motifs in the MS-MS data. Potential applications envisaged for SALSA include identification of specific protein modifications, e.g., PTM, identifications of peptides with common sequences, e.g., wild-type and mutant forms, and targeted analysis of isoforms and conformers in complex samples. 

Novel methods for the direct mass spectral analysis of breath samples have been reported. Most of these detect volatile components but one has used extractive electrospray ionisation MS to directly detect non-volatile lipids, peptides and carbohydrates as well as volatile compounds in the breath of subjects. ... 

Figure 4a MALDI-TOF mass spectral analysis of H-Thr-Arg-Asn-Leu-Ala-Asp-Gln-Glu-Asp-NH2. The spectrum was acquired on a TofSpec SE (Micromass) mass spectrometer equipped with a nitrogen laser and a reflectron, using a peptide solution of approximately 10 pmol//u,L and HCCA (Hewlett-Packard) as the matrix.

Figure 4b ESI mass spectral analysis of H-Thr-Arg-Asn-Leu-Ala-Asp-Gln-Glu-Asp-NH2. The spectrum was acquired on a Finnigan LCQ ion trap mass spectrometer the spectrum obtained by mathematical deconvolution is shown in the inset. The sample was dissolved at a concentration of approximately 0.5 pmol//xL in 50% acetonitrile containing 0.5% acetic acid and introduced into the mass spectrometer by direct infusion at a flow rate of 5 iL/min. Spectral averaging of 20 scans was employed. The small peaks in the spectrum were produced by in-source CAD of the peptide peaks such as these are frequently, but not always observed in ESI-MS analysis of small peptides.

Second, the positions and llneshapes of resonances arising from potentially mobile parts of the peptide (e,g, side chains) have revealed dynamical aspects of the solid-state structures of peptides. The analysis of molecular motions is simplified In the solid state by the absence of overall molecular tumbling, which modulates spin interactions and leads to complex frequency -dependent spectral responses. In particular, signals arising from aromatic ring side chains are well separated from other resonances, and may be interpreted in terms of reorientation models of these side chains. Such ring dynamics are of great importance in protein structures, and studies with model peptides can help elucidate fundamental aspects of these processes. 

Pnor to automated sequencing or mass spectrometry, one may wish to concentrate the peptide in a somewhat smaller tube Transfer the sample to a 1500 or 500 uiL Eppendorf polypropylene tube and combine with a rinse of the 12 x 75 mm tube (rinse with 100 pL of 80% MeCN/0.01% TFA it is advisable to rinse the tube several times if practicable). Immediately remove the solvent by vacuum centrifugation. We advise using only the transparent (uncolored) Eppendorf tubes to handle peptide solutions. Do not use tubes of any kind with O rings or septum closures because they have been shown to contain compounds that can interfere with mass spectral analysis. If a microbore HPLC system is available,... 

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